Open AccessSpecific interaction between a Saccharomyces cerevisiae protein and a DNA element associated with certain autonomously replicating sequences.
Author(s) -
Shlomo Eisenberg,
Chris Civalier,
Bik Kwoon Tye
Publication year - 1988
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.85.3.743
Subject(s) - biology , saccharomyces cerevisiae , dna , dna replication , autonomously replicating sequence , tandem affinity purification , base pair , deoxyribonuclease i , binding site , dna binding site , oligonucleotide , plasmid , hmg box , dna binding protein , origin of replication , dna footprinting , microbiology and biotechnology , footprinting , genetics , biochemistry , yeast , gene , transcription factor , affinity chromatography , enzyme , promoter , gene expression , base sequence
We have isolated a protein from Saccharomyces cerevisiae that binds specifically to a nucleotide sequence associated with the autonomously replicating sequence (ARS) ARS120, located in the telomeric region of a yeast chromosome. "Footprinting" analysis revealed that a 26-base-pair DNA sequence, 5'-CAAGTGCCGTGCATAATGATGTGGGT-3', was protected by this protein from DNase I digestion. A plasmid containing 48 direct tandem repeats of this oligonucleotide was constructed and used to affinity-purify the binding activity. The purified protein, OBF1 (origin binding factor), showed specific binding to ARS120. The 26-base-pair OBF1-protected sequence was sufficient for the recognition and binding of the protein, since the mobility of a DNA fragment containing the synthetic binding site was retarded in agarose gels when incubated with OBF1. By performing competition experiments with a number of different ARSs, we showed that OBF1 binds tightly to some but not all ARSs. Interestingly, OBF1 does not appear to have a discernible affinity for ARS1 or the ARSs associated with mating type loci, HML alpha and HMRa, which are substrates for a DNA-binding activity reported by others. Since OBF1 appears to bind to DNA associated with a number of ARSs, we suggest that this protein may have a function related to ARS activity, perhaps in the initiation of DNA replication at selected ARSs.